In vitro, the mitogenic stimulation of human T-lymphocytes usually favors the proliferation of gamma/delta-T cells but can often induce apoptosis in gamma/delta-T cells, thus preventing their expansion. We have identified a CD2-mediated, interleukin (IL)-12-dependent signaling pathway which inhibits apoptosis in mitogen-stimulated human gamma/delta-T cells. Biologically, these findings are significant as they may contribute to a greater understanding of how this or similar signaling pathways serve to maintain or regulate the gamma/delta-T cell compartment in vivo. Moreover, these findings have an important practical significance: By exploiting this signaling pathway, our laboratory has been able to develop the methodologies permitting the large-scale in vitro expansion of viable, apoptosis-resistant human gamma/delta-T cells, an undertaking until now, not possible. As importantly, these apoptosis-resistant gamma/delta-T cells (now readily obtainable) can be shown to mediate significant innate, major histocompatibility complex (MHC)-unrestricted cytotoxicity against a variety of human tumor cell lines in vitro, including melanoma cell lines which were found to be exquisitely sensitive to cytolysis mediated by apoptosis-resistant gamma/delta-T cells, but not control alpha/beta-T cells. In this grant, two distinct, yet related questions regarding apoptosis-resistant gamma/delta-T cells will be addressed: 1) What are the biological mechanisms underlying the enhanced expansion and survival of apoptosis-resistant gamma/delta-T cells? and, 2) How might we begin to exploit for direct clinical benefit the intrinsic or innate antitumor properties displayed by apoptosis-resistant gamma/delta-T cells? The first question can be restated as our testable overall hypothesis which is that: CD2-mediated, IL-12-dependent signaling is a mechanism by which human gamma/delta-T cells can acquire resistance to apoptosis and that this pathway is an important determinant in the regulation of the survival (and therefore, function) of gamma/delta-T cells. This basic question will be addressed primarily in Specific Aim 1 where we propose to examine the molecular and cellular consequences of CD2-mediated, IL-12-dependent signaling in human gamma/delta-T cells with respect to inhibition of apoptosis. The second question relates to a corollary to our overall hypothesis which is that: Apoptosis-resistant gamma/delta-T cells which readily expand in culture - yet retain intrinsic or innate MHC-unrestricted antitumor cytotoxicity - can provide an important means to examine both the pre-clinical biology and the direct clinical potential of apoptosis-resistant human gamma/delta-T cells as a new form of adoptive cellular immunotherapy for melanoma. This question will be addressed in Specific Aim 2 and Specific Aim 3 where we will define the biology of the antitumor cytotoxicity mediated by apoptosis-resistant gamma/delta-T cells against human melanoma cells. A key feature to these studies is that we will examine the innate antitumor activity of apoptosis-resistant gamma/delta-T cells derived from both normal individuals and from patients with melanoma. Ultimately, we will directly determine whether melanoma-reactive apoptosis-resistant gamma/delta-T cells can be identified and propagated in vitro from both peripheral blood and from primary tumor samples (gamma/delta-T cell tumor-infiltrating lymphocytes, or TIL) obtained from patients with melanoma. These findings may provide the basis for the rational design of future immunotherapy trials for the treatment of melanoma or other malignancies.